Steven A Rosenberg and Cancer Immunotherapy

Steven A Rosenberg MD and the Race for CAR T-Cell Cancer Therapy

Laboring tediously for hours on end, medical students inject cancer cells into mice, producing many publications in prestigious journals. As you might expect, all the cancer injected mice died promptly from cancer. All except one. The unexpected happened. This one mouse survived, and was renamed, “the mouse that killed cancer.” As Luis Pasteur once said, Fortune favors the prepared mind. Above Left Image Steven A Rosenberg MD PhD courtesy of OncLive.

Spontaneous Regression of Cancer in the Mouse
Once identified as a “cancer killing” mouse, the little furry fellow was earmarked for study. These were exciting times, and there were many questions. Why didn’t this mouse die of cancer like all the others? How was this mouse able to defeat the injected cancer cells? What kind of immune system protected this mouse? Left Image: Laboratory Mouse courtesy of wikimedia commons.

A Mouse Immune to Cancer

Over the next 3 years, studies showed this strain of mice had an innate immunity to cancer, a genetic trait passed on to the offspring. Their T-Cells, T-lymphocytes, recognize and kill cancer cells, just as if cancer was an invading microorganism. These mice were called SR/CR mice, which stands for Spontaneous Regression/Complete Resistant to Cancer.(2)

Saving Other Mice From Cancer

What about the other plain mice with no immunity to cancer who quickly succumb to injected cancer cells? Could these plain mice be protected from cancer by transferring the immune system from a SR/CR mouse? What if we infused the T-Cells, the immune cells from a SR/CR mouse into a plain mouse?

More experiments showed yes, this is correct. Protection from the injected cancer cells could be transferred to plain mice after transfusion with T-Cells from SR/CR mice.(3) In addition, the protective SR/CR T-Cells still retained activity after many weeks of cold storage.(4)

Human Mice – Spontaneous Regression of Cancer

What about us humans? Do we have similar immunity to cancer? Are some of us humans “immune” from cancer? The answer is “Yes”, and this is called spontaneous remission of cancer, reported many times in the medical literature. Spontaneous remission/regression has been reported for neuroblastoma, renal cell carcinoma, malignant melanoma and lymphomas/leukemias (see Papac RJ and Chodorowski Z). (5)(6)

The Legend of Sir William Osler – Spontaneous Regression

In 1901, the legendary Dr William Osler, reported spontaneous remission in fourteen cases of breast cancer . (reference: The Medical Aspects of Carcinoma of the Breast, Spontaneous Disappearance of Secondary Growths OSLER W., American Medicine: April 6 1901; 17-19; 63-66.)

Spontaneous Regression in Breast Cancer

A study by Dr Gilbert Welch concluded that small breast cancers may spontaneously regress as reported by Gina Kolata in the New York Times . Spontaneous regression in the cancer patient is a victory of the immune system over the malignant potential of the cancer mass. Left Image Sir William Osler 1912 courtesy of wikimedia.

Adoptive Immunotherapy – Dr Steven A Rosenberg NIH

Inspired by this SR/CR Immune Mouse in which immunity to cancer may be transferred from one mouse to another, Steven A Rosenberg MD at the National Cancer Institute, developed a cancer treatment using T-Cells infused into cancer patients. He called this “Adoptive Cell Transfer Immunotherapy”, and results have been remarkable.(7)

Dr Rosenberg ‘s treatment involves pre-treating the patient’s T lymphocytes to increase the anti-tumor activity. The patient’s blood is drawn, the lymphocytes isolated, and then activated and cloned in a test tube. These activated lymphocytes are then re-infused into the patient. This method is very effective for metastatic melanoma, producing tumor regression in 50% of patients in clinical trials. See Rosenberg’s case images showing tumor regression (7). See Figure 2 from his article showing examples of tumor regression in patients receiving immune cells, lymphocytes (white cells).(7)

Enter the CAR T Cell, Chimeric Antigen Receptor Engineered T-Cell

What if we could re-engineer the patients T-Cells, the immune cells, to recognize and attack surface antigens on the cancer cell? Using modern biotechnology techniques this can be done. The technique is called CAR, which stands for Chimeric Antigen Receptor.(8-12) See left diagram of CAR T-Cell courtesy of The Pharmaceutical Journal .

CD19 Marker on B-Cells

The most promising efforts using CAR T Cells have been directed at the CD19 marker on B-Cell malignancies such as Leukemia and Lymphoma. These B cell cancers have the CD19 surface antigen marker allowing the CAR T-Cells to recognize and destroy them.(8-12)

As of 2014, there have been 13 publications of clinical trials using CAR T cells for treatment of B-cell malignancies targeting the CD19 or CD20 surface antigen. These have, showing dramatic remissions in a number of patients with refractory B-cell lymphoma. (10)(12) The CAR technique has also been tried in neuroblastoma, and sarcoma patients. (11)

Low Dose Chemotherapy Followed by CAR

Dr Steven Rosenberg co-authored an abstract at the June 2016 ASCO meeting, presenting 22 patients with B cell malignancies treated with low dose chemotherapy followed by anti-CD19 CAR engineered T-Cells.(13) Most of these cases were refractory to chemotherapy or had relapsed after autologous stem cell transplant (these are stem cells harvested from the patient and re-infused). Following treatment, about half of the patients had ongoing complete remissions, an impressive result.(13)

CAR added to Allogeneic Transplant Protocol

Dr Steven Rosenberg and Jennifer Brudno reported in 2016 using anti-CD19 engineered T cells as part of an allogeneic transplant protocol.(14) Twenty lymphoma patients with progressive disease after allogeneic stem cell transplant were given anti CD-19 T-Cells prepared from the transplant donor. Results were favorable with the most impressive remissions in ALL (acute lymphoblastic leukemia). There was no graft-versus-host disease and toxicities were acceptable. The authors predicted that in the near future, CAR engineered T Cells will be routinely added to allogeneic stem cell transplant protocols. (14,15)

Combination Therapies Improve Efficacy of CAR T Therapy

The dramatic results of CAR T in pediatric lymphoblastic leukemia have been difficult to achieve in adult B cell lymphoma cases. Thus, a search for combination therapies is underway to enhance the efficacy of CAR T therapy. For example, combination therapy with the BTK inhibitor, Ibrutinib, added to CAR T Cell therapy has been found to improve efficacy and reduce cytokine storm.(35)(39) Combination therapy with a COX 2 inhibitor, such as Celecoxib (Celebrex), and/or the BCL2 inhibitor, Venetoclax, both increase efficacy of CAR-T Cell therapy.(36-38)

Three biotech companies, Juno, Kite and Novartis, are in clinical trials racing towards FDA approval for their CAR T-Call product lines. The treatment of B cell cancers has been successful because B Cells are the only ones expressing CD19 marker, and also because we can live without B cells. The first CAR T-cell therapy for B-Cell cancers could enter the market in 2017.(17) Left Image foot race courtesy NY Times.

According to Rachel Webster: “since 2014, CAR T-cell therapies have achieved impressive complete response (CR) rates of up to 90% in pediatric and adult patients with relapsed or refractory (R/R) acute lymphocytic leukemia (ALL) and non-Hodgkin’s lymphoma (NHL); durable remissions and six-month survival of more than 70% have been reported.” (17)

Making CAR T-Cell Therapy More Effective: Ibrutinib

Results of CAR T Cell Therapy for Non-Hodgins Lymphoma can be improved with co-administration of Ibrutinib, (Brutons Tyrosine Kinase Inhibotor). Studies by Steven Schuster at U Penn using mouse – mantle cell lymphoma xenografts show more durable long term remission when ibrutinib is co-adminsisterd with the CAR T cell therapy. (28)

According to Dr Wasik in a 2015 report, Mantle Cell Lymphoma cells have CB1 Cannabinoid receptors which are not present on normal lymphocytes, and found in the central nervous system, perhaps explaining ready attachment to nervous tissue. For prevention or treatment of nervous system lymphoma, Ibrutinib is an excellent choice, since it readily crosses the blood brain barrier, and showed impressive results in three cases mantle cell lymphoma with CNS relapse reported in 2015 by Dr Sophie Bernard.in Paris.(32) “All three patients had dramatic and rapid responses with almost immediate recovery from CNS symptoms. “(32)

Cytokine Release Syndrome

An adverse side effect of CAR T-Cell therapy is cytokine release syndrome (CRS), the release of inflammatory cytokines upon interaction of the CAR T cells with the malignant cells. This adverse effect seems most pronounced in cases with heavy disease burden, highlighting the need for cytoreductive chemotherapy prior to T Cell infusion. “Severe (and potentially fatal) CRS has been observed in approximately one-third of R/R ALL patients.”(17) Another potential adverse effect is long lasting loss of the B-cell population which puts the patient at risk for infections. (16)

A Treatment with Rock-Star Status Suffers SetBack

In July 2016, the FDA temporarily halted Juno’s CAR-T clinical trials at Memorial Sloan Kettering Cancer Center in New York after three patient deaths due to cerebral edema from cytokine-release syndrome (CRS),(18) They quickly resumed enrollment after a short one week delay. Juno’s CAR T technology is patented by Memorial Sloan Kettering Cancer Center in New York (MSK).

Wounded Juno Allows KITE to FLY

As Juno falls behind, KITE captures the lead with their KTE-619 CAR T-Cell product. In June 2016, Kite opened their 43,000 square foot manufacturing facility for commercial production of KTE-619 CAR-T Cells, with capacity to treat 5,000 patients per year. The new plant will be operational by Dec 2016 Turnaround time from lymphopheresis to reinfusion of the KTE C19 product is approximately 2 weeks, one of the fastest in the industry.(19) Shawn Tomasello of KITE says he is “building out a commercial team that will target 50 to 70 cancer centers.” (20)

Novartis CAR T Given FDA Approval

August 30, 2017, Novartis working in collaboration with Dr. Carl June at the Perelman center at the University of Pennsylvania is the first CAR T Cell therapy, Kymriah(TM)(tisagenlecleucel) (CTL019) , to receive FDA approval for pediatric B-Cell Lymphoblastic Leukemia (relapsed/refractory). “Oxford BioMedica is the sole manufacturer of the lentiviral vector that encodes CTL019.”(40-41)

FDA Approval given for Yescarta (axicabtagene ciloleucel), CAR T Cell therapy developed by KITE and now owned by Gilead, to treat adult patients with Relapsed/Refractory Large B-cell lymphoma. “In a multicenter clinical trial of more than 100 adults with refractory or relapsed large B-cell lymphoma. The complete remission rate after treatment with Yescarta was 51 percent.”(42)

CD19 Negative Relapse After CD19 CAR T Cell Therapy

Although early clinical trial data shows high complete remission rate in B Cell hematologic malignancies, there are cases which relapse with loss of CD19 surface antigen. Explanations for relapse with loss of CD19 vary. Dr Inga Nagel from Kiel Germany working with a bispecific antibody, blinatumomab, says in Blood 2017 this is “due toselection of preexisting CD19-negative malignant progenitor cells”. (59) Other labs at MD Anderson and Rutgers going back to 2010 found that progenitor cells, also called cancer stem cells, in B Cell hematologic malignancies were CD19 negative, while the bulk of the tumor mass contained CD19 positive cells. (54-58) These CD19 negative progenitor cells are highly chemotherapy resistant and explain the rapid relapse after treatment in the highly proliferation B cell malignancies. If this is true, then targeting the CD19 marker may provide tumor debulking and complete remissions, admittedly an advantage in the Relapsed/Refractory setting. However, the CD19 negative progenitor cells will remain behind to cause relapsed disease after a time interval determined by cell proliferation rate.

UPenn Data on CD19 CAR-T for Pediatric Lympho Blastic Leukemia

Although initial impressive results were obtained with 93% complete remissions after CTL019 infusion in 59 children at UPenn for pediatric lymphoblastic leukemia, after one year, 45% of the chuildren had relapsed, with 13 cases of CD19- (negative).(60) Indeed the high relapse rate has been identified as a major problem.(61). This type of human data supports the hypothesis that CD19- (negative) cancer stem cells evade treatment, and initiate relapse.(60-64)

The use of dual marker CAR T (CD19 and CD22) has been explored to over-come CD19 relapse problems. Interest has expanded to other markers such as the BCMA (Juno/MSK Clinical Trial), with preliminary data looking very good in multiple myeloma.

Roaring Ahead with ROR CAR T Juno CAR 024

Both the University of California at San Diego, and Seattle based JUNO in collaboration with Fred Hutch Cancer center are developing the ROR CAR T (Receptor Orphan Receptor 1) now in clinical trial JCAR 024.(43-44) Principal Investigator for JCAR024 is David Maloney at Fred Hutch/University of Washington.(44) The ROR surface antigen is closely associated with the WNT pathway, and uniformly expressed at high levels in Mantle cell Lymphoma and CLL.(45) . Inhibition of the WNT pathway targets the cancer stem cells, so the ROR targeted agents may actually eradicate cancer stem cells providing a “cure”.(45) We anxiously await longer term follow up data on this.

Conclusion: One can only stand in awe of Dr Steven A Rosenberg and his almost herculean accomplishments in the field of cancer immunotherapy ultimately bearing fruit as CAR T-cell Therapy for B-Cell Malignancies. Of the three biotech companies now in a race for FDA approval, KITE seems poised to grasp the prize.

We have established and studied a colony of mice with a unique trait of host resistance to both ascites and solid cancers induced by transplantable cells. One dramatic manifestation of this trait is age-dependent spontaneous regression of advanced cancers. This powerful resistance segregates as a single-locus dominant trait, is independent of tumor burden, and is effective against cell lines from multiple types of cancer. During spontaneous regression or immediately after exposure, cancer cells provoke a massive infiltration of host leukocytes, which form aggregates and rosettes with tumor cells. The cytolytic destruction of cancer cells by innate leukocytes is rapid and specific without apparent damage to normal cells. The mice are healthy and cancer-free and have a normal life span. These observations suggest a previously unrecognized mechanism of immune surveillance, which may have potential for therapy or prevention of cancer.

Amy M. Hicks et al. The killing of cancer cells in SR/CR mice requires three distinct phases. First, the leukocytes must migrate to the site of cancer cells after sensing their presence. Second, they must recognize the unique properties of the cancer cell surface and make tight contact with it. Third, the effector mechanisms must finally be delivered to target cells. The difference between SR/CR and WT mice seems to lie in one of the first two phases. Upon challenge with cancer cells, WT mice lack leukocyte infiltration and rosette formation. Apparently, the mutation in SR/CR mice renders the leukocytes capable of sensing unique diffusible and surface signals from cancer cells, and of responding to the activation signals by migration and physical contact. Once the first two phases are accomplished, unleashing the pre-existing effector mechanisms for killing seems to ensue by default. Therefore, the mutated gene (or genes) likely determines whether leukocytes interpret the signals from cancer cells as inhibition, as in WT leukocytes, or as activation of migration and target recognition, as in SR/CR leukocytes. Identifying the mutated gene (or genes) will likely explain this unique resistance to cancer through immunity.

Abstract Background
Spontaneous Regression/Complete Resistant (SR/CR) mice are resistant to cancer through a mechanism that is mediated entirely by leukocytes of innate immunity. Transfer of leukocytes from SR/CR mice can confer cancer resistance in wild-type (WT) recipients in both preventative and therapeutic settings. In the current studies, we investigated factors that may impact the efficacy and functionality of SR/CR donor leukocytes in recipients.

Spontaneous regression of cancer is reported in virtually all types of human cancer, although the greatest number of cases are reported in patients with neuroblastoma, renal cell carcinoma, malignant melanoma and lymhomas/leukemias. Study of patients with these diseases has provided most of the data regarding mechanisms of spontaneous regression. Mechanisms proposed for spontaneous regression of human cancer include: immune mediation, tumor inhibition by growth factors and/or cytokines, induction of differentiation, hormonal mediation, elimination of a carcinogen, tumor necrosis and/or angiogenesis inhibition, psychologic factors, apoptosis and epigenetic mechanisms. Clinical observations and laboratory studies support these concepts to a variable extent. The induction of spontaneous regression may involve multiple mechanisms in some cases although the end result is likely to be either differentiation or cell death. Elucidation of the process of spontaneous regression offers the possibility of improved methods of treating and preventing cancer.

Spontaneous regression of malignant tumours is a rare and enigmatic phenomenon. We reviewed the cases of spontaneous regression of cancer in medical literature according to MEDLINE database in the period 1988-2006 and compared them with similar reviews from 1900-1987 period. The number of reported cases of spontaneous regression increased steadily in XX century, probably due to a rising interest in this problem and new possibilities of radiological and biopsy examinations. Spontaneous regression of malignancy was reported in almost all types of human cancer, although the greatest number of cases in years 1988-2006 were reported in patients with nephroblastoma, renal cell carcinoma, malignant melanoma, lymphoma. Elucidation of the process of spontaneous regression offers the possibility of improved methods of preventing andlor treating cancer.

Adoptive cell therapy (ACT) using autologous tumour-infiltrating lymphocytes has emerged as the most effective treatment for patients with metastatic melanoma and can mediate objective cancer regression in approximately 50% of patients. The use of donor lymphocytes for ACT is an effective treatment for immunosuppressed patients who develop post-transplant lymphomas. The ability to genetically engineer human lymphocytes and use them to mediate cancer regression in patients, which has recently been demonstrated, has opened possibilities for the extension of ACT immunotherapy to patients with a wide variety of cancer types and is a promising new approach to cancer treatment.

Figure 2
Examples of objective tumour regressions in patients receiving adoptive cell transfer of autologous anti-tumour lymphocytes following a lymphodepleting preparative regimen
In each case the pretreatment scans and photos are shown on the left and the post-treatment on the right. a | A 45-year-old male with metastatic melanoma to the liver (upper) and right adrenal gland (middle) who was refractory to prior treatment with high dose α interferon as well as high-dose interleukin 2 (IL2). He underwent a rapid regression of metastases and developed vitiligo (lower). b | A 55-year-old male with rapid tumour growth in the axilla as well as multiple brain metastases from metastatic melanoma that was refractory to prior treatment with high dose IL2 who underwent rapid regression of nodal and brain metastases.

The future of ACT

In contrast to common epithelial cancers, melanoma appears to be a tumour that naturally gives rise to anti-tumour T cells. However, other cancers are equally susceptible as the targets of reactive T cells. The susceptibility of melanoma to ACT provides optimism for the application of ACT to common epithelial cancers using TCR gene-modified lymphocytes.

A major problem with the application of ACT is that it is a highly personalized treatment and does not easily fit into current modes of oncological practice. The treatment is labour-intensive and requires laboratory expertise. In essence, a new reagent is created for each patient and this patient-specific nature of the treatment makes it difficult to commercialize. Pharmaceutical and biotechnology companies seek off-the-shelf drugs, easy to produce, vial and administer. From a regulatory standpoint, ACT might be more appropriately delivered as a service rather than as a ‘drug’. Blood banks have been instrumental in providing CD34+ haematopoietic stem cells for clinical studies and might be the ideal location for the generation of the anti-tumour T cells needed for ACT.

As modern science increasingly provides the physician with sophisticated information about the unique aspects of an individual cancer, changes in the modes of care delivery need to accommodate this. The ability to use this patient-specific information can lead to a new era of personalized medicine in which individual treatments, such as ACT, are devised for each patient.Studies of ACT have clearly demonstrated that the administration of highly avid anti-tumour T cells directed against a suitable target can mediate the regression of large, vascularized, metastatic cancers in humans and provide guiding principles as well as encouragement for the further development of immunotherapy for the treatment of patients with cancer.

8) Setting the Body’s ‘Serial Killers’ Loose on Cancer By ANDREW POLLACK AUG. 1, 2016 New York Times Dr. Steven Rosenberg, who turns 76 on Tuesday, still works nearly every day. “I want to end this holocaust,” he said of cancer. Chimeric antigen receptor (CAR) T-cell therapy

11) Dai, Hanren, et al. “Chimeric antigen receptors modified T-cells for cancer therapy.” Journal of the National Cancer Institute 108.7 (2016): djv439. The genetic modification and characterization of T-cells with chimeric antigen receptors (CARs) allow functionally distinct T-cell subsets to recognize specific tumor cells. The incorporation of costimulatory molecules or cytokines can enable engineered T-cells to eliminate tumor cells. CARs are generated by fusing the antigen-binding region of a monoclonal antibody (mAb) or other ligand to membrane-spanning and intracellular-signaling domains. They have recently shown clinical benefit in patients treated with CD19-directed autologous T-cells. Recent successes suggest that the modification of T-cells with CARs could be a powerful approach for developing safe and effective cancer therapeutics.
Adoptive immunotherapy for cancer has a long and somewhat checkered history; the first observations that immune system engagement had antitumor effects are commonly attributed to William Coley, who observed the regression of sarcoma following severe bacterial infections in the 1890s (1). However, the seminal finding that hematopoietic stem cell transplantation (HSCT) using syngeneic donors (from identical twin) was less effective at preventing relapse of leukemia compared with sibling donors provided the founding rationale for adoptive T-cell therapy (2). Additionally, the direct isolation and ex vivo activation of the tumor-infiltrating lymphocytes (TILs) was tested in multiple early-phase studies and resulted in durable responses in melanoma (3).
Recently, laboratory studies of chimeric antigen receptor (CAR)–specific T-cells have been viewed with exceptional interest for clinical development at an array of academic institutions. The redirection of T-cells to tumor antigens by expressing transgenic chimeric antigen receptors takes advantage of potent cellular effector mechanisms via human leukocyte antigen (HLA)–independent recognition. The potential of this approach has recently been demonstrated in clinical trials, wherein T-cells expressing CAR were infused into adult and pediatric patients with B-cell malignancies, neuroblastoma, and sarcoma (4–12).

12) free pdf
Almåsbak, Hilde, Tanja Aarvak, and Mohan C. Vemuri. “CAR T Cell Therapy: A Game Changer in Cancer Treatment.” Journal of Immunology Research 2016 (2016). Almåsbak Hilde CAR T Cell Therapy Game Changer Cancer Immunology Research 2016
The development of novel targeted therapies with acceptable safety profiles is critical to successful cancer outcomes with better survival rates. Immunotherapy offers promising opportunities with the potential to induce sustained remissions in patients with refractory disease. Recent dramatic clinical responses in trials with gene modified T cells expressing chimeric antigen receptors (CARs) in B-cell malignancies have generated great enthusiasm. This therapy might pave the way for a potential paradigm shift
in the way we treat refractory or relapsed cancers. CARs are genetically engineered receptors that combine the specific binding domains from a tumor targeting antibody with T cell signaling domains to allow specifically targeted antibody redirected T cell activation. Despite current successes in hematological cancers, we are only in the beginning of exploring the powerful potential of CAR redirected T cells in the control and elimination of resistant, metastatic, or recurrent nonhematological cancers. This review
discusses the application of the CAR T cell therapy, its challenges, and strategies for successful clinical and commercial translation.

Years of successive and significant innovations have finally culminated in clinical studies demonstrating the tremendous potential of second generation CAR expressing T cells (Figure 1). Genetic redirection of patient T cells with
CARs targeting the B lymphocyte antigen CD19 has met with exceptional success in various therapy-refractory hematologic diseases (reviewed in [9]). Given their remarkable activity, CAR T cells are expected to enter the mainstream of health care for refractory or relapsed B-cell malignancies within few years and become the game changer for similar approaches in treating other cancers, such as solid tumors.

Background: T cells genetically-modified to express chimeric antigen receptors (CARs) targeting CD19 have potent activity against a variety of B-cell malignancies. Chemotherapy is administered prior to CAR T cells because depletion of recipient leukocytes enhances the anti-malignancy efficacy of adoptively-transferred T cells; an increase in serum interleukin (IL)-15 is one mechanism for this enhancement. Previously, we (Kochenderfer et al. Journal of Clinical Oncology, 2015) and others have reported patients treated with high-dose chemotherapy prior to anti-CD19 CAR T-cell infusions. This report describes treatment of 22 patients with low-dose conditioning chemotherapy followed by infusion of anti-CD19 CAR T-cells. Methods: Eighteen of 22 treated patients received 300 mg/m2 of cyclophosphamide (cy) daily for 3 days; 4 patients received 500 mg/m2 of cy on the same schedule. All patients received fludarabine 30 mg/m2daily for 3 days on the same days as cy. Patients received a single dose of CAR T cells 2 days after completion of chemotherapy. Blood CAR T cells and serum cytokines were analyzed in all patients. Results: Nineteen patients with various subtypes of diffuse large B-cell lymphoma (DLBCL) had the following responses: 8 CR, 5 PR, 2 SD, and 4 PD. One patient with mantle cell lymphoma obtained a CR.Two patients with follicular lymphoma both obtained CRs. Durations of response currently range from 1 to 20 months; 10 remissions are ongoing. All but 4 patients had either chemotherapy-refractory lymphoma or lymphoma that had relapsed after autologous stem cell transplant. The most prominent toxicities were various neurological toxicities. Other toxicities included fever and hypotension. The median peak blood CAR+ cell level was 47/μL (range 4-1217/μL). Patients obtaining CRs or PRs had higher peak blood CAR+ cell levels than patients experiencing SD or PD. The mean serum IL-15 level was 4 pg/mL before the conditioning chemotherapy and 32 pg/mL after chemotherapy (P < 0.0001). Conclusions: Anti-CD19 CAR T cells can induce remissions of advanced B-cell lymphoma when administered after low-dose chemotherapy. In the near future, CAR T cells will likely be a standard therapy for lymphoma.

Remarkable early-phase clinical data for CAR T-cell therapy was first announced in 2014, and since then excitement has been growing. CAR T-cell therapies have achieved impressive complete response (CR) rates of up to 90% in pediatric and adult patients with relapsed or refractory (R/R)acute lymphocytic leukemia (ALL) and non-Hodgkin’s lymphoma (NHL); durable remissions and six-month survival of more than 70% have been reported.
Off-target sides effects associated with CAR T-cell therapy, namely cytokine release syndrome (CRS), are a major concern. Severe (and potentially fatal) CRS has been observed in approximately one-third of R/R ALL patients. A high-risk of CRS could confine CAR T-cell approaches to late-line treatment settings, when other treatments have been exhausted.
The first CAR T-cell therapy could enter the market in 2017.

18) Rock star cancer treatment is being scrutinized after clinical trial deaths
Jamie Reno, Yahoo Finance Contributor July 31, 2016
In 2014, Juno’s CAR-T clinical trials were stopped temporarily at Memorial Sloan Kettering Cancer Center in New York due to several patient deaths as a result of cytokine-release syndrome (CRS), a potentially deadly toxicity associated with increased levels of cytokines in the body including interleukin (IL)-6 and interferon γ.

19) UPDATED: Racing past a wounded Juno, Kite aims to file lead CAR-T for OK by end of 2016 by John Carroll August 8, 2016
Less than two months ago, Kite held a ribbon cutting ceremony for its new manufacturing facility, a 43,500-square-foot plant that will be used to make its personalized KTE-C19.
“Our commercial facility will have the capacity to produce up to 5,000 patient therapies per year and we expect it to be operational in producing clinical materials by year-end,” noted Belldegrun. “Overall, we have continuously been optimizing key aspect of our manufacturing, supply chain, and quality control and possess a proprietary process that dramatically reduces the time to approximately 14 days for when a patients material are shift to our facility to when the engineered T-cells are released to the patient. This is one of the fastest rates in the industry.”

If Kite achieves this goal, it will position itself to win approval for KTE-C19 as a treatment for diffuse large B-cell lymphoma, primary mediastinal B cell lymphoma and transformed follicular lymphoma next year. With the brief clinical hold causing Juno’s targeted approval to slip from 2017 to 2018, Kite is positioned to come to market well before its rival, while also pipping Novartis ($NVS) to the post.
Asked how soon after approval Kite would be ready to launch KTE-C19, Chief Commercial Officer Shawn Tomasello was unequivocal.
“Immediately. We’ll be ready,” Tomasello said.
To live up to Tomasello’s bullishness, Kite will need to have all its commercial and logistic units ready to go by some time next year. Work in these areas is already well underway. Tomasello is in the middle of building out a commercial team that will target 50 to 70 cancer centers.

21) T-cell therapies for cancer: from outsider to pharmaceutical darling
The Pharmaceutical Journal 24 AUG 2016 By Janna Lawrence
there are now three competing companies. And all of them are working on cell therapies that target CD19 on B cells.
The treatment of B cell cancers has been so successful because they are the only cells that express CD19 and also because a person can live without B cells.

1Center for Cellular Immunotherapies, University of Pennsylvania, Philadelphia, PA 2Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 3Center for Chronic Lymphocytic Leukemia, Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA 4Division of Hematology-Oncology, University of Pennsylvania, Philadelphia, PA
5Pathology and Laboratory Medicine, Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA
Introduction: The bruton tyrosine kinase (BTK) inhibitor ibrutinib demonstrates considerable activity in mantle cell lymphoma (MCL). However, approximately 30% of patients do not respond to this treatment and the therapy invariably leads to drug resistance with a median response of 17.5 months. Infusion of autologous T cells transduced with chimeric antigen receptors (CAR) against the B-cell specific CD19 antigen (CART19) leads to dramatic clinical responses in the majority of patients with acute lymphoblastic leukemia and the activity in B cell lymphoma is currently being evaluated in clinical trials. Bulky disease, as sometimes seen in MCL, may impair T cell infiltration. The features of ibrutinib that make it an interesting addition to CART19 include its efficacy in reducing tumor masses and its ability to mobilize neoplastic B cells into the peripheral blood, thereby potentially exposing them to the killing activity of CART19. Therefore, we sought to investigate the combination of the two novel targeted therapies, ibrutinib and CART19 in MCL.

Results: In vitro studies with established MCL cell lines and with a novel cell line (MCL-RL) showed a range of responses to ibrutinib with an IC50 ranging from 10 nM to 10 µM; MCL-RL was the most sensitive cell line evaluated with an IC50 of 10nM, similar to primary MCL. Both ibrutinib-sensitive and ibrutinib-resistant cell lines strongly activated CART19 in an antigen-specific manner as detected by CD107a degranulation, cytokine production and CFSE proliferation assays. Importantly, in vitro assays with MCL cell lines co-cultured with increasing doses of CART19 (E:T= 2:1, 1:1, 0.5:1, 0.25:1) combined with increasing concentrations of ibrutinib (0, 10, 100, 1000 nM) demonstrated strong additive tumor killing (Figure 1). Notably, supra-therapeutic doses of Ibrutinib (>/=1 uM) impaired cytokine production and T cell proliferation in vitro. In order to test this combination in vivo we established a novel MCL model, injecting i.v. luciferase-positive MCL-RL cells into NSG mice. This resulted in 100% MCL engraftment in liver and spleen, with eventual dissemination into lymph nodes and bone marrow. Treatment with three different doses of CART19 (0.5, 1 and 2 million cells/mouse) led to a dose dependent anti-tumor effect. A similar dose response to CART19 was also observed in the ibrutinib-resistant Jeko-1 cell line. We also treated MCL-RL xenografts with different doses (0, 25 and 125 mg/Kg/day) of ibrutinib, with a median overall survival respectively of 70, 81 and 100 days (p<0.001). Importantly, a direct in vivo comparison of the highest ibrutinib dose (125 mg/kg) and CART19 showed a significantly improved tumor control for mice treated with CART19. However, treatment with either CART19 or ibrutinib as single agents invariably led to late relapse. Therefore we sought to treat MCL-RL xenografts with the combination of CART19 and ibrutinib and compare it to the single agent activity. The combination resulted in significant improvement in tumor control compared to mice treated with the single agents with 80% of mice achieving long-term disease-free survival (p=0.007 at day 110, representative mice shown in Figure 2A). Intriguingly, we found that mice treated with ibrutinib had higher numbers of circulating CART19 cells (Figure 2B).

Conclusions: Combining CART19 with ibrutinib represents a rational way to incorporate two of the most recent therapies in MCL. Our findings pave the way to a two-pronged therapeutic strategy in patients with MCL and other types of B-cell lymphoma.

The field of adoptive cell transfer (ACT) is currently comprised of chimeric Ag receptor (CAR)- and TCR-engineered T cells and has emerged from principles of basic immunology to paradigm-shifting clinical immunotherapy. ACT of T cells engineered to express artificial receptors that target cells of choice is an exciting new approach for cancer, and it holds equal promise for chronic infection and autoimmunity. Using principles of synthetic biology, advances in immunology, and genetic engineering have made it possible to generate human T cells that display desired specificities and enhanced functionalities. Clinical trials in patients with advanced B cell leukemias and lymphomas treated with CD19-specific CAR T cells have induced durable remissions in adults and children. The prospects for the widespread availability of engineered T cells have changed dramatically given the recent entry of the pharmaceutical industry to this arena. In this overview, we discuss some of the challenges and opportunities that face the field of ACT.

However, up to one third of MCL patients do not respond to ibrutinib and among the responders only a third achieve complete remission (CR).

Using in vitro and in vivo models of MCL, including a novel cell line highly sensitive to ibrutinib, we demonstrate here that CTL019 is more effective than ibrutinib as monotherapy, and that the addition of ibrutinib to CTL019 further augments the anti-tumor effect and leads to prolonged remissions.’

The nonsteroidal anti-inflammatory drug (NSAID) Celecoxib (Celebrex®) received Food and Drug Administration (FDA) approval in 1998 for treatment of osteoarthritis and rheumatoid arthritis, and in recent years, its use has been extended to various types of malignancies, such as breast, colon, and urinary cancers. To maintain the survival of malignant B cells, non-Hodgkin’s Lymphoma (NHL) is highly dependent on inflammatory microenvironment,
and is inhibited by celecoxib. Celecoxib hinders tumor growth interacting with various apoptotic genes, such as cyclooxygenase-2 (Cox-2), B-cell lymphoma 2 (Bcl-2) family, phosphor-inositide-3 kinase/serine-threonine-specific protein kinase (PI3K/Akt), and inhibitors of apoptosis proteins (IAP) family. CD19-redirected chimeric antigenreceptor
(CD19 CAR) T cell therapy has shown promise in the treatment of B cell malignancies. Considering its regulatory effect on apoptotic gene products in various tumor types, Celecoxib is a promising drug to be used in combination with CD19 CAR T cell therapy to optimize immunotherapy of NHL.

B-cell malignancies upregulate the B-cell lymphoma 2 (Bcl-2) family inhibitors of the intrinsic apoptosis pathway, making them therapy resistant. However, small-molecule inhibitors of Bcl-2 family members such as ABT-737 restore a functional apoptosis pathway in cancer cells, and its oral analog ABT-263 (Navitoclax) has entered clinical trials.

Gene engineered chimeric antigen receptor (CAR) T cells also show promise in B-cell malignancy, and as they induce apoptosis via the extrinsic pathway, we hypothesized that small-molecule inhibitors of the Bcl-2 family may potentiate the efficacy of CAR T cells by engaging both apoptosis pathways.

CAR T cells targeting CD19 were generated from healthy donors as well as from pre-B-ALL (precursor-B acute lymphoblastic leukemia) patients and tested together with ABT-737 to evaluate apoptosis induction in five B-cell tumor cell lines.

The CAR T cells were effective even if the cell lines exhibited different apoptosis resistance profiles, as shown by analyzing the expression of apoptosis inhibitors by PCR and western blot. When combining T-cell and ABT-737 therapy simultaneously, or with ABT-737 as a presensitizer, tumor cell apoptosis was significantly increased.In conclusion, the apoptosis inducer ABT-737 enhanced the efficacy of CAR T cells and could be an interesting drug candidate to potentiate T-cell therapy.

Chimaeric antigen receptor (CAR) T-cells have shown impressive results in patients with B-cell leukaemia.Yet, in patients with lymphoma durable responses are still rare and heavy preconditioning required. Apoptosis resistance is considered a hallmark of cancer, often conveyed by a halted apoptosis signalling. Tumours regularly skew the balance of the components of the apoptotic machinery either through up-regulating anti-apoptotic proteins or silencing pro-apoptotic ones. Malignant B-cells frequently up-regulate anti-apoptotic B-cell lymphoma 2 (Bcl-2) family proteins leading to therapy resistance. CAR T-cells kill tumour cells via apoptosis induction and their efficacy may be affected by the level of Bcl-2 family proteins. Hence, there is an interesting possibility to increase the effect of CAR T-cell therapy by combining it with apoptosis inhibitor blockade agents. Compounds that inhibit Bcl-2, B-cell lymphoma extra large (Bcl-xL) and Bcl-2-like protein 2 (Bcl-w), can restore execution of apoptosis in tumour cells or sensitize them to other apoptosis-dependent treatments. Hence, there is a great interest to combine such agents with CAR T-cell therapy to potentiate the effect of CAR T-cell killing. This review will focus on the potential of targeting the apoptotic machinery to sensitize tumour cells to CAR T-cell killing.

ALTHOUGH CHIMERIC ANTIGEN RECEPTOR (CAR)-T CELLS as a treatment for B-cell neoplasms have shown some promising results in clinical trials, their clinical use is limited, partially due to the risk of cytokine-release syndrome (CRS) occurring in response to the treatment. A poster presented at the Annual Meeting of the American Society of Hematology demonstrated that mice receiving CAR-T immunotherapy plus ibrutinib demonstrated longer overall survival and reduced cytokine production than the mice not treated with ibrutinib.1

“Cytokine-release syndrome is a serious adverse event of anti-CD19 chimeric antigen receptor T-cell (CART19) therapy and could potentially limit its widespread clinical use,” explained lead study author Marco Ruella, MD, clinical instructor at the Perelman School of Medicine Center for Cellular Immunotherapies at the University of Pennsylvania. “In this preclinical study, we demonstrated that the [Bruton’s tyrosine kinase]-inhibitor ibrutinib administered with CART19 can modulate cytokine production by CAR T cells and neoplastic B cells, therefore reducing CRS and increasing survival.”

They created a human xenograft of CRS by infusing CART19 cells into mice that had a high B-cell tumor burden. The mice began to display signs of distress resembling CRS, including reduced mobility and hyperventilation, 2 days after the injection. Compared with the controls, CART19-treated mice showed significantly higher serum concentrations of several human cytokines. The researchers then tested their hypothesis that ibrutinib would reduce CART19-mediated CRS without impairing the anti-tumor efficacy of these cells.2

Ibrutinib, an inhibitor of Bruton’s tyrosine kinase, has been approved as a first-line treatment for chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). It has been shown to modulate T-cell cytokine production, and the researchers recently demonstrated that its combination with CART19 leads to enhanced antitumor responses in preclinical models of MCL, CLL, and B-cell acute lymphoblastic leukemia.

To test their hypothesis, the researchers administered either a combination of CART19 plus ibrutinib or CART19 alone to mice with a high tumor burden of MCL. Mice treated with the ibrutinib combination demonstrated prolonged overall survival (median 17.5 days) compared with the mice that received the CART19 alone (median 5 days). Serum measurements 4 days after treatment showed that the ibrutinib-treated mice had significantly reduced cytokines, including IL-6, IFN?, TNFa, IL-2, and GM-CSF.

“In vitro studies revealed that ibrutinib reduced cytokine production by CAR-T cells, as well as by MCL cells, leading us to postulate that both CRS and its successful prevention involve cross-talk between immune cells and cancer cells,” the study authors wrote. They suggested that the CART19/ ibrutinib combination “could be a novel strategy” in preventing CRS.
REFERENCES

Public Abstract: Autologous ROR1 CAR-T cell transduced with a lentiviral vector containing scFv (cirmtuzumab) with CD28, CD137, CD3zeta signaling domains
Area of Impact ROR1 expressing cancer stem cells in solid tumors and hematologic tumors
Mechanism of Action ROR1 CAR is a 3rd generation chimeric construct with an internal endodomain that transmits a CD3 zeta signal with added co-stimulatory signaling domains 4-1BB and CD28. When the transduced ROR-1 CAR-T cell comes into contact with its cognate receptor, a signal is transmitted by the CD3 zeta-chain, inducing lymphocyte proliferation and expression of trans-acting interleukins and chemokines that activate other immuno-reactive cells and in certain cases directly kill ROR1+ Cancer Stem Cells

Compelling evidence suggests that dormant cancer stem cells (CSCs) are considered the origin of therapeutic resistance, and are responsible for relapse and metastasis. We will selectively identify and attack CSCs through the ROR1 receptor, using CAR-T cells to address this unmet medical need.

Functional data suggest that ROR1 may act in Wnt-signaling and promote the survival of malignant cells.16,17 Here, we characterize the expression of ROR1 in B-cell malignancies and human tissues, and show that in addition to B-CLL, ROR1 is expressed uniformly at high levels in MCL and transiently at a specific stage of normal B-cell development but not in major adult tissues. CD8+ T cells engineered to express a ROR1-specific CAR selectively lyse primary B-CLL and MCL, but not normal mature B cells in vitro, suggesting that ROR1-specific T-cell therapy may be an effective treatment for patients with ROR1-positive B-cell tumors. Uniform high-level expression of ROR1 on MCL was confirmed in 6/6 primary clinical samples. A potential advantage of targeting ROR1 rather than B-cell lineage-specific molecules such as CD20 or CD19 is that ROR1 is not expressed on mature normal B cells.

Clinical-stage biotechnology company Oncternal Therapeutics announced the closing of an $18.4 million Series B financing. The company which develops first-in-class and novel therapies for both rare and common cancers by focusing on targets that are uniquely expressed within cancer cells, intends to use the proceeds to further clinical development programs for cirmtuzumab and TK216, and to advance preclinical development of a new ROR1-targeted antibody-drug conjugate or ADC program.

The Receptor Tyrosine Kinase-Like Orphan Receptor 1 or ROR1 is an oncofetal protein which has gained much attention in cancer therapy since its initial discovery as a relatively specific surface antigen on B cell chronic lymphocytic leukemia (CLL). [1] Researchers have confirmed that ROR1 mediates several oncogenic pathways in a cancer type- and context-dependent manner. They have also found that ROR1 is mainly expressed in cells during embryogenesis and is re-expressed in a growing number of cancer types including, among others, malignant melanoma, breast cancer, and prostate cancer. [2][3]

According to the latest 2016 data from the National Cancer Institute an estimated 8,960 people were diagnosed with Chronic lymphocytic leukemia or CLL in the United States and 4,660 died of the disease, which is characterized by reposition of malignant B cells in the blood, bone marrow, spleen and lymph nodes. Over the last few years major breakthroughs have been made to prolong the survival and improve the health of patients. However, despite these advances, CLL is still recognized as a disease without definitive cure.

Cirmtuzumab

Cirmtuzumab, also known as UC-961, is a humanized IgG1 monoclonal antibody designed and developed to bind with high affinity to a biologically important epitope on the extracellular domain of ROR1. As a first-in-class anti-ROR1 monoclonal antibody being developed to treat patients with chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). ROR1 expression identifies cancer stem cells in a number of hematologic malignancies and solid tumors, and that certain antibodies against ROR1 inhibit malignant behavior. Initial findings from the ongoing Phase Ia clinical trial in patients with relapsed or refractory CLL, including signs of pharmacological activity and prolonged progression-free survival, were presented at the American Society of Hematology (ASH) Meeting in December 2016. [4]

The investigational drug cirmtuzumab was developed at the University of California San Diego based on the pioneering scientific research of Thomas Kipps, M.D. Ph.D, and his colleagues at the Moores Cancer Center.Oncternal holds an exclusive worldwide license to develop and commercialize antibodies and antibody-related binding agents recognizing ROR1.

Ets-family transcription factor oncoproteins

Oncternal is also developing another drug. Called TK216, this investigational agent is a first-in-class small molecule that inhibits the biological activity of ets-family transcription factor oncoproteins in a variety of tumor types, inhibiting cancer cell growth and tumor formation in nonclinical models. In Ewing sarcoma, TK216 is designed to target a fusion protein that causes the disease. A Phase I clinical trial in patients with relapsed or refractory Ewing sarcoma is currently underway. Oncternal has received Orphan Drug Designation and Fast Track status from the U.S. Food and Drug Administration (FDA) for TK216 in Ewing sarcoma and will be eligible to receive a Rare Pediatric Disease Priority Review Voucher if approved for this indication. [5]

Antibody-drug conjugate

Oncternal is also investigating the potential of an ROR1-targeted antibody-drug conjugates. Preclinical models show that upon binding, ROR1 antibodies such as cirmtuzumab are rapidly internalized and traffic inside the cell in a manner that is ideal for delivering a cytotoxic payload. Based on this understanding, Oncternal is generating and evaluating a series of ROR1-targeted antibody-drug conjugates utilizing several different toxic payloads, linkers and conjugation chemistry. Preclinical evaluation of these candidates will continue during 2017.

“We are extremely pleased with the rapid progress we have made in our clinical development programs since Oncternal was formed just nine months ago, and we appreciate the strong support of our shareholders and new investors,” said James Breitmeyer, M.D., Ph.D., Oncternal’s President and CEO. “We have reached the top dose group in our Phase 1a clinical trial of cirmtuzumab without safety issues and with evidence of pharmacological activity. We are preparing to launch a Phase Ib/II clinical trial of cirmtuzumab combined with ibrutinib as treatment for patients with CLL and MCL. With TK216, we have progressed through four dose levels in our Phase Ia study for patients with Ewing sarcoma without dose limiting toxicity, and have new nonclinical data to support development of TK216 for patients with hematologic malignancies. Additionally, we have initiated an anti-ROR1 ADC program and are testing product candidates from the program to add to our development pipeline.”

Adult patients who are interested in T cell therapies at Penn Medicine can call 215-316-5127 for more information. For information about the Cancer Immunotherapy Program at CHOP, please call 267-426-0762.

Here we report that MCL-initiating activities are enriched in a subpopulation of tumor cells that lack the prototypic B cell marker, CD19. As few as 100 CD45+CD19- MCL cells displayed self-renewal activities and formed tumors in immunodeficient mice. In contrast, CD45+CD19+ MCL cells were not able to self-renew during serial transplantation assays and displayed greatly reduced tumorigenicity.

MCL-initiating cells (MCL-ICs) have been recently identified based on a lack of CD19 marker (CD34-CD3-CD45+CD19- cells) [25]. Two studies from different groups have shown that these MCL-ICs can repopulate tumor in mice [25, 26]. As few as 100 of CD19- MCL-ICs have been found to produce whole tumor with both CD19+ and CD19- cells, while CD19+ MCL-non-ICs were incapable of tumor development at comparable limited dilutions in severe combined immunodeficiency (SCID) mice [25, 26]. We suggest that the high relapse rates of human MCL arise from incomplete elimination of chemoresistant MCL-Cs

The expression levels of the antioxidant enzymes MT1b and SOD2 were elevated over sixfold in MCL-ICs, suggesting a higher reactive oxygen species scavenging capacity (Fig. 2b). MCL-ICs also overexpressed genes associated with chemoresistance, such as those encoding the ATP transporters ABCC3 and ABCC6 as well as CD44 (>100-, 22-, and 3-fold, respectively) compared with MCL-non-ICs (Fig. 2c).

Burton tyrosine kinase (BTK) has been shown to be a negative regulator of Wnt signaling [29]. Therefore, it is not surprising that ibrutinib (a BTK inhibitor) probably resulted in inducing Wnt signaling rather than inhibiting it and thereby could not eliminate MCL-ICs. Our results suggest that the inability of conventional chemotherapy to kill MCL-ICs can be overcome by adding inhibitors of Wnt signaling.

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We discovered that CD45+CD19- MCL cells, which we termed MCL-initiating cells (MCL-ICs), are highly tumorigenic and display self-renewal capacity in vivo; in contrast, CD45+CD19+ MCL cells, which constitute the vast majority of cells within the tumors, show no self-renewal capacity and greatly reduced tumorigenicity.

We recently identified clonogenic malignant stem cell populations in human mantle cell lymphoma (MCL), a particularly deadly subtype of non-Hodgkin lymphoma (NHL). We discovered that CD45+CD19- MCL cells, which we termed MCL-initiating cells (MCL-ICs), are highly tumorigenic and display self-renewal capacity in vivo; in contrast, CD45+CD19+ MCL cells, which constitute the vast majority of cells within the tumors, show no self-renewal capacity and greatly reduced tumorigenicity. Given the newly appreciated role of cancer-initiating cells in the drug resistance of cancers, it is critical to investigate whether CD45+CD19- MCL-ICs play a role in the drug resistance of human MCL. We discovered that MCL-ICs were more resistant to clinically relevant chemotherapeutic agents, in combination or in a single regimen, compared to CD45+CD19+ cells, and that this drug resistance was largely due to quiescent properties with enriched ABC transporters. In conclusion, designing novel therapies to kill CD45+CD19- MCL-ICs may prevent relapse and increase patient survival.

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In this report we present experiments that demonstrate that only this CD19-CD133+ subpopulation of primary MCL cells can self renew and engraft immunodeficient mice.

Mantle cell lymphoma (MCL) is associated with a significant risk of therapeutic failure and disease relapse, but the biological origin of relapse is poorly understood. Here, we prospectively identify subpopulations of primary MCL cells with different biologic and immunophenotypic features. Using a simple culture system, we demonstrate that a subset of primary MCL cells co-cultured with either primary human mesenchymal stromal cells (hMSC) or murine MS-5 cells form in cobblestone-areas consisting of cells with a primitive immunophenotype (CD19-CD133+) containing the chromosomal translocation t (11;14)(q13;q32) characteristic of MCL. Limiting dilution serial transplantation experiments utilizing immunodeficient mice revealed that primary MCL engraftment was only observed when either unsorted or CD19-CD133+ cells were utilized. No engraftment was seen using the CD19+CD133- subpopulation. Our results establish that primary CD19-CD133+ MCL cells are a functionally distinct subpopulation of primary MCL cells enriched for MCL-initiating activity in immunodeficient mice. This rare subpopulation of MCL-initiating cells may play an important role in the pathogenesis of MCL.

Analysis of this CAFC MCL cell population showed that these clusters contain self-renewing cells with the chromosomal translocation t(11;14)(q13;q32) characteristic of MCL. Yet these cells have a unique immunophenotype, namely the expression of the HSC marker CD133 and loss of CD19 expression. In this report we present experiments that demonstrate that only this CD19-CD133+ subpopulation of primary MCL cells can self renew and engraft immunodeficient mice.

Mantle cell lymphoma (MCL) is an aggressive and incurable form of non-Hodgkin’s lymphoma. Despite initial responses to intense chemotherapy, up to 50% of cases of MCL relapse, often in a chemoresistant form. We hypothesized that the recently identified MCL-initiating cells (MCL-ICs) are the main reason for relapse and chemoresistance of MCL.

Methods

We isolated MCL-ICs from primary MCL cells on the basis of a defined marker expression pattern; CD34-CD3-CD45+CD19-. The MCL-ICs, MCL-non-ICs, and peripheral blood lymphocytes from healthy donors were analyzed for gene expression using the Arraystar platform. The differences in mRNA levels of genes of interest were confirmed by quantitative RT-PCR. The prominent differentially expressed transcripts were analyzed using the Ingenuity Platform. Primary MCL cells were co-culture with mesenchymal stem cells to assess the effects of chemotherapeutic agents such as vincristine, doxorubicin and the newly approved Burton tyrosine kinase inhibitor ibrutinib, and Wnt signaling inhibitors.

Results

Approximately 1% of primary MCL cells are MCL-ICs and they can be maintained in co-culture with mesenchymal stem cells. Comparison of gene expression profiles of MCL-ICs and MCL-non-ICs revealed activation of stem cell-specific pathways in MCL-ICs by expression of Wnt, Notch, and Hedgehog and enhanced expression of Nanog, Oct4, KLF4, ADH1, MT1b and ABCC3. Gene expression microarray data and RT-PCR data suggested predominant activation of the Wnt/Frizzled pathway. Indeed, MCL-ICs were particularly sensitive to Wnt pathway inhibitors. Targeting Wnt-dependent ß-catenin?TCF4 interaction with CCT036477, iCRT14, or PKF118-310 preferentially eliminated the MCL-ICs, reduced the expression of stem cell transcription factors (Myc, Nanog, Oct4, Klf4), and sensitized MCL cells to vincristine, doxorubicin, and ibrutinib. Interestingly, while vincristine, doxorubicin or ibrutinib did kill MCL cells, they did not reduce the percentage of MCL-ICs in treated co-culture.

Conclusion: MCL-ICs are present in primary MCL isolates and they show gene expression pattern of chemoresistant, stem cell-like cells with predominant activation of Wnt signaling. In order to produce durable remissions in MCL patients, treatment strategies should be directed to target MCL-ICs.

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Here, we provide evidence that CD19-negative relapse after CD19-directed therapy in BCP-ALL may be due to selection of preexisting CD19-negative malignant progenitor cells. Bi-Directional Antibody.

The bispecific T-cell engager blinatumomab targeting CD19 can induce complete remission in relapsed or refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, some patients ultimately relapse with loss of CD19-antigen on leukemic cells which has been established as a novel escape mechanism to CD19-specific immunotherapies. Here, we provide evidence that CD19-negative relapse after CD19-directed therapy in BCP-ALL may be due toselection of preexisting CD19-negative malignant progenitor cells. We present two BCR-ABL1-fusion-positive BCP-ALL patients with CD19-negative myeloid lineage relapse after blinatumomab therapy and show BCR-ABL1-positivity in their hematopoietic stem cell (HSC)/progenitor/myeloid compartments at initial diagnosis by fluorescence in situ hybridization after cell sorting. Using the same approach in 25 additional diagnostic samples of patients with BCR-ABL1-positive BCP-ALL, HSC involvement was identified in 40% of the patients. Patients with major-BCR-ABL1 transcript encoding P210BCR-ABL1 mainly showed HSC involvement (6/8), whereas in most of the patients with minor-BCR-ABL1 transcript encoding P190BCR-ABL1 only the CD19-positive leukemia compartments were BCR-ABL1-positive (9/12) (p=0.02). Our data are of clinical importance, because they indicate that not only CD19-positive cells, but also CD19-negative precursors should be targeted to avoid CD19-negative relapses in patients with BCR-ABL1-positive ALL.

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